Several experimental models have failed to demonstrate significant regeneration in the lesioned adult spinal cord.sup.1-5. Few neurons in the spinal cord regenerate even when provided with a permissive substrate. Analysis of transplantation experiments.sup.6-10 demonstrates that less than 1% of the neurons within the potential pool of neurons regenerate into the transplant. This suggests that promotion of significant regeneration in the spinal cord requires activation of the growth program. Neurotrophins promote growth and differentiation of neurons and have been shown to be involved in both the development and maintenance of the central nervous system (CNS).sup.11-13. They are expressed in similar patterns in both developing and regenerating neurons and also rescue neurons following axotomy.sup.14-16.
Nerve Growth Factor (NGF) is a neurotrophic (NT) factor.sup.17. More than two decades after the discovery of NGF, brain-derived neurotrophic factor (BDNF) was isolated from brain tissue.sup.18. Using sequence homologies between NGF and BDNF, neurotrophin-3 (NT3).sup.19,20 and neurotrophin-4 (NT-4) (also called NT-5 or NT-4/5).sup.21 were subsequently identified and characterized. NT factors mediate their biological effect by binding with high affinity to cell surface glycoprotein receptors. These receptors are encoded by the trk family of protooncogenes. Trk receptor proteins contain an extracellular ligand binding domain and an intracellular tyrosine kinase domain. The extracellular ligand binding domain activates the intracellular tyrosine kinase domain. NGF binds to the trk gene product, gp 140 .sup.trk (trkA).sup.22,23. BDNF and NT-4 bind to the two glycoproteins, gp95.sup.trk B and gp145.sup.trB (trkB), which are encoded by the trkB gene.sup.24-26. gp95.sup.trk B is a truncated form of the trkB receptor molecule and lacks the tyrosine kinase domain.sup.27. NT-3 binds to gp.sup.145trkC (trkC) and also, with less affinity, to trkA and trkB.sup.28. The trkC gene locus also codes an isoform lacking the intracellular catalytic domain.sup.28. Additionally, all the NTs bind with low affinity to a transmembrane glycoprotein, p75.sup.NGFR (p75).sup.29. The levels of trkA in the CNS are extremely low, and are concentrated to basal forebrain cholinergic neurons that are dependent on NGF.sup.30. Full-length trkB and trkC are expressed during the development of the CNS, and are down-regulated to barely detectable levels in the adult.sup.31.
There is some evidence that neurons in the adult spinal cord and projection neurons to the spinal cord respond to BDNF and NT-3. BDNF and NT-3 treatment increased the number of regenerating axons 2-fold in Schwann cell seeded silicon tubes implanted into the midthoracic spinal cord.sup.32. Retrograde tracing showed that these neurons (92 per animal) were scattered throughout the rostral cord and into the brain stem. Schwab and colleagues showed that NT-3 enhances sprouting of corticospinal tract after lesion in the adult spinal cord. Rubrospinal projection neurons respond to BDNF following axotomy by upregulating GAP-43.sup.33. BDNF prevents neurotoxin-induced loss of 5-HT neurons and promotes sprouting of uninjured 5-HT axons.sup.34. Infusion of either BDNF or NT-3 significantly elevated the antinociceptive response in rat.sup.35. This response is mediated by descending 5-HT fibers.
There are changes in the expression of trkB following lesions in the spinal cord, however, only for transcripts of truncated trkB, which are increased throughout the scar in both rats and cats.sup.36,37. There is no change in either full-length trkB or trkC both in the scar and in the surrounding spinal cord tissue. The few axons which had regenerated into the scar were associated with the glial cells which express truncated trkB. Sciatic nerve or dorsal root lesions also upregulate truncated trkB in the spinal cord.sup.38. This upregulation in the scar or denervated tissue in the adult spinal cord could contribute to regeneration.
During the period of neurogenesis in the spinal cord of rat, both BDNF and NT-3 and their full-length catalytic receptors are upregulated.sup.39-41. Transcripts for both full-length trkB and trkC are expressed at low levels on embryonic day 13 (E-13) and progressively increase during the period of rapid neuronal growth and differentiation. Expression of trkB catalytic form in the CNS peaks on postnatal day 1 (P1) and progressively decreases to barely detectable levels in the adult; full-length trkC similarly peaks at P1 and decreases to levels comparable to E13 levels in the adult. Transcripts for the truncated trkB receptors also peak shortly after birth, and, by contrast, remain at relatively high levels in the adult. The level of p75.sup.NGFR transcripts is 5-fold higher at E13-14 than P1, and it also declines to barely detectable levels in the adult spinal cord. A similar pattern has been reported in developing spinal cord of chicks.sup.39. This developmental pattern of early expression of full-length trkB and trkC and significantly later expression of truncated trkB is common in wide variety of regions of the CNS and indicates that the regulation of the ratio of full-length to truncated NT receptors plays an important role in the development and maintenance of the CNS.sup.39,40.
Consistent with this developmental pattern, neurons which regenerate or sprout following a lesion in the adult nervous system have upregulation of full-length trkB. Accompanying this upregulation is the upregulation of truncated receptors in glial cells associated with the neurite substrate.
The first example illustrating these changes is granule cell axons of the dentate gyrus; they sprout after seizures induced by intraventricular kainic acid injections or by electrolytic lesions in the hippocampus.sup.42. Full-length trkB mRNA and proteins are upregulated dramatically within 5 hours post-lesion over the cell bodies of granule cells. BDNF levels increase 30-fold, and NT-3 levels decreased over the same time course.sup.42. There are no changes in trkA or trkC mRNA expression.
Correlated with the above changes is the upregulation of GAP-43.sup.43 which is a good marker for neurite outgrowth. Ischemic and hypoglycemic conditions result in similar changes in the hippocampus.sup.44. Further, mRNAs for BDNF and trkB are coexpressed in hippocampal neurons which suggests an autocrine mechanism.sup.45. Neurotrophins are also changed in the dentate gyrus following deafferentation (fornix-fimbria and perforant path lesions). Beck and colleagues.sup.46 observed pronounced increase in truncated trkB over glial cells which match, in time and place, to the region into which sprouting of axons occurs. They speculate that "noncatalytic trkB molecules expressed on the surface of glial cells play an important role in plasticity of the adult brain, possibly regulating the concentration of bioactive neurotrophin or the responsiveness of neurotrophin receptors".
Another neuronal type in the adult nervous system which has upregulation of neurotrophin system following a lesion and during regeneration are neurons in dorsal root ganglia (DRG). BDNF as well as NGF are expressed in adult DRG neurons, and following sciatic nerve lesions, both are upregulated by 2- and 3-fold respectively.sup.47. Both full-length trkB and trkC levels are also increased (2 times and slightly, respectively), and p75 levels are transiently depressed and return to normal levels within 1 week.sup.38,47. Accompanying this is the upregulation of truncated trkB and trkC expression which is confined to Schwann cells.sup.37,48. These observations again suggest that the noncatalytic forms of the trkB and trkC as well as the p75.sup.NGFR might recruit or present the NT factors to the full-length receptors present on the regenerating axons.sup.37,39,49. The similarities between developing and regenerating neurons with respect to neurotrophic factors and their receptors suggest that these factors play a role in regeneration.
In summary, lesions in the spinal cord upregulate truncated forms of trkB in glial cells, but there are no changes in the full-length trkB associated with neurons. The presence of full-length trkB and trkC in most developing CNS neurons and in the neurons shown to regenerate in the adult nervous system (including the peripheral nervous system (PNS) led to the conclusion that upregulation of full-length catalytic trkB and trkC in neurons in the injured central nervous system will induce regeneration of axons and promote functional recovery.
An object of the present invention is to provide methods that result in survival and/or regeneration of neurons.
Another object of the present invention is to provide methods to induce expression of (upregulate) neurotrophic factor receptors trkB and trkC in neurons of the adult nervous system.
According to certain embodiments, the present invention provides methods for treating patients with nervous system damage, including but not limited to CNS trauma and strokes (e.g., spinal cord injury). According to certain embodiments the present invention provides methods for treating neurodegenerative disorders, including by not limited to, Alzheimer's disease, Parkinson's disease, Huntington's chorea, and amyotrophic lateral sclerosis and other motor neuropathies.
The present inventors have shown that treatment of adult spinal cord with the combination of all-trans retinoic acid (RA), dibutyryl cyclic AMP (dBcAMP) and KCl induces the expression of functional forms of trkB and trkC. The present inventors have also shown that such treatment is not damaging to the spinal cord and can be applied to treatment of neurotrauma, stroke, and neurodegenerative diseases. Without being limited to any theory of why such treatments work, the present inventors believe that the treatment results in upregulation of functional forms of trkB and trkC. Accordingly, the present methods can be used for conditions in which such upregulation is typically needed for functional recovery.